Ana Jiménez

Estrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human Neutrophils

Abstract —Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17β-estradiol (10−10 to 10−8 mol/L) upregulated the expression of nNOS protein. The 17β-estradiol receptor antagonists, tamoxifen (10−8 mol/L) and ICI 182780 (10−8 mol/L), inhibited the upregulation of nNOS protein induced by 17β-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17β-estradiol–incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17β-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17β-estradiol–incubated neutrophils derived from men to produce NO reduced their adhesive properties.

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor‐α release by cultured smooth muscle cells

Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS).

Losartan inhibits in vitro platelet activation: comparison with candesartan and valsartan.

A recent study has shown that losartan, an AT1-receptor antagonist, interacts with thromboxane A2 (TxA2)/prostaglandin H2 (PGH2) receptors in human platelets. The aim of the present study was to analyse the ability of different angiotensin II (Ang II) AT1-receptor antagonists to inhibit TxA2-dependent human platelet activation. Platelets were obtained from healthy volunteers and were stimulated with the thromboxane A2 analogue, U46619 (10-6 mol/L). U46619-stimulated platelet activation was significantly reduced by losartan in a dose-dependent manner. Only maximal doses of valsartan (5x10-6 mol/L), reduced U46619-induced platelet activation. The active form of candesartan cilexetil, candesartan (CV-11974), failed to modify platelet activation. Losartan reduced the binding of [3H]-U46619 to platelets, an effect that was observed to a lesser extent with valsartan but not with CV-11974. These results suggest that, whilst some AT1-receptor antagonists reduce TxA2-dependent human platelet activation, it is not a feature common to all AT1 antagonists.

Inhibition of platelet activation in stroke-prone spontaneously hypertensive rats: comparison of losartan, candesartan, and valsartan.

In vitro studies have suggested that losartan interacts with the thromboxane (TxA2)/ prostaglandin H2 (PGH2) receptor in human platelets, reducing TxA2-dependent platelet activation. The aim of this study was to evaluate the effect of different angiotensin II type 1 receptor antagonists in stroke-prone spontaneously hypertensive rats (SHRSP). The level of platelet activation was assessed by determining P-selectin expression in platelets by flow cytometry. The ex vivo adhesion of platelets was also analyzed. The number of platelets that expressed P-selectin in SPSHR was significantly increased (% P-selectin expression: WKY 4 ± 0, 4%; SHRSP 15.5 ± 0, 8% [n = 8], p < 0.05). In SHRSP receiving losartan (20 mg/kg body weight per day) the percentage of platelets expressing P-selectin fell to levels close to that observed in WKY. The number of platelets from SHRSP treated with valsartan and candesartan (20 mg/kg body weight per day for 14 days) that expressed P-selectin was not significantly different from those from untreated SPRHR. Only losartan treatment reduced ex vivo platelet adhesion to a synthetic surface. The antiplatelet effect of losartan does not appear to be related to the level of blood pressure reduction. In ex vivo experiments, losartan significantly reduced the binding of the radiolabeled TxA2 agonist U46619 to platelets obtained from SHRSP in a dose-dependent manner. Treatment with losartan reduced the number of activated platelets in SHRSP independently of its blood pressure effects. TxA2-receptor blockade is proposed as a mechanism by which losartan can prevent platelet activation.

Aspirin Prevents Escherichia coli Lipopolysaccharide– and Staphylococcus aureus–Induced Downregulation of Endothelial Nitric Oxide Synthase Expression in Guinea Pig Pericardial Tissue

The aim was to analyze whether pericardial tissue expresses endothelial NO synthase (eNOS) protein and to determine the presence of cytosolic proteins that bind to eNOS mRNA. The effect of aspirin on the above-mentioned parameters was also analyzed. eNOS protein was expressed in pericardial tissue from male guinea pigs. Escherichia coli lipopolysaccharide (LPS, 10 &mgr;g/mL) and Staphylococcus aureus endotoxin (SA, 10 &mgr;g/mL) reduced eNOS protein expression and shortened the half-life of the eNOS messenger. Under basal conditions, cytosolic extracts from pericardial samples bound to the 3′-untranslated region (3′-UTR) of eNOS mRNA, which was enhanced by LPS and SA. Proteinase K fully prevented the binding of cytosolic pericardial extracts to 3′-UTR of eNOS mRNA, suggesting the involvement of proteins that were further characterized as 60- and 51-kDa proteins. Aspirin (1 to 10 mmol/L) restored eNOS expression in either LPS- and SA-stimulated pericardial samples and reduced the binding activity of the pericardial cytosolic proteins to 3′-UTR of eNOS mRNA. Indomethacin also reduced the downregulation of eNOS by LPS and diminished the binding activity of the cytosolic proteins, although higher doses of indomethacin than of aspirin were needed to improve these parameters. In conclusion, eNOS protein is expressed in guinea pig pericardial tissue. LPS and SA stimulate the binding activity of pericardial cytosolic proteins to 3′-UTR of eNOS mRNA and reduce eNOS protein expression. High doses of aspirin and indomethacin protect eNOS protein expression and reduce the binding activity of the cytosolic proteins to 3′-UTR of eNOS mRNA, suggesting an inverse association between the presence of these cytosolic proteins and eNOS expression.

Efecto de la inhibición de la HMG-CoA reductasa sobre la proteína inductora de disfunción endotelial en conejos hipercolesterolémicos☆

Introduction and objectives. In our laboratory, we recently obtained evidence that cultured bovine endothelial cells contain cytosolic proteins that form complexes with the 3’-unstranslated region of endothelial nitric oxide synthase (eNOS) mRNA and are associated with its destabilization. The aim of this study was to determine the presence of such proteins and the level of eNOS expression in hypercholesterolemic rabbits as an in vivo model of endothelial dysfunction. Methods and results. Endothelium-dependent relaxation in response to acetylcholine was reduced in aortic segments from hypercholesterolemic rabbits compared with controls. Treatment of hypercholesterolemic rabbits with simvastatin (25 mg/kg body weight/day) restored endothelium-dependent relaxation. Aortic eNOS expression was reduced in hypercholesterolemic rabbits and was accompanied by enhanced binding activity of a 60-KDa cytosolic protein and reduced stability of eNOS mRNA. Simvastatin treatment upregulated eNOS expression and reduced the interaction of cytosolic protein with the 3’-untranslated region of eNOS mRNA. Conclusions. These results demonstrate the presence of a 60-KDa protein that binds to eNOS mRNA and reduces eNOS expression in the vascular wall.

Efecto del triflusal sobre la agregación y secreción de las plaquetas humanas: papel del óxido nítrico

INTRODUCTION AND AIMS: The thrombotic process is a multicellular phenomenon in which not only platelets are involved but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal reduced platelet aggregation by stimulating nitric oxide (NO) production by neutrophils. The aim of the present study was to evaluate whether the in vivo treatment with triflusal could also modify the ability of neutrophils to produce NO. Furthermore, the role of NO released by neutrophils on platelet aggregation and secretion was also tested. METHODS: The study was performed in 12 healthy volunteers of 32 +/- 6 years of age. The volunteers were treated with triflusal (600 mg/day) for 5 days and platelets and neutrophils were isolated before and after treatment. The ability of neutrophils to produce NO and the capacity of inhibiting platelet aggregation and secretion of transforming growth factor-beta (TGF-beta) were assessed. RESULTS: After the treatment with triflusal we obtained the following results: a) an increase in NO production by neutrophils; b) potentiation of the inhibition of platelet aggregation by neutrophils, an effect that was reverted by incubating neutrophils with an L-arginine antagonist, L-NAME, and c) the presence of neutrophils reduced the release of TGF-beta by platelets measured as index of platelet secretion by a NO-independent mechanism. CONCLUSIONS: Triflusal (600 mg/day/5 days) stimulated NO production by neutrophils. After the treatment with triflusal, neutrophils inhibited both platelet aggregation and secretion. The antiaggregating effect of neutrophils was an NO-dependent mechanism while the inhibition of platelet secretion mediated by neutrophils after the treatment with triflusal was an NO-independent mechanism.

Efecto del losartán sobre la activación de plaquetas humanas por tromboxano A2

Introduccion y objetivo Estudios previos han demostrado que el losartan, antagonista de los receptores de tipo AT-1 de la angiotensina II (Ang II) podria bloquear al receptor del tromboxano A2 (TXA2) en la pared vascular. El objetivo del trabajo fue estudiar el efecto del losartan sobre la activacion de plaquetas humanas. Materiales y metodos Las plaquetas fueron obtenidas de 15 voluntarios sanos con edades comprendidas entre los 26 y 40 anos. La activacion plaquetaria fue medida por cambios en la transmision de luz del plasma rico en plaquetas estimuladas por un analogo sintetico del TXA2, el U46619 (5 × 10 –6 mol/l). Resultados El U46619 estimulo la agregacion de las plaquetas, siendo significativamente inhibida por el losartan de manera dosis dependiente. Solo dosis altas del EXP 3174, el metabolito hepatico principal del losartan, consiguieron inhibir la activacion plaquetaria inducida por el U46619. Captopril, inhibidor de la enzima convertidora de angiotensina, no fue efectivo en modificar la agregacion plaquetaria inducida por el analogo del TXA2. A pesar de que las plaquetas expresan receptores de tipo AT-1 de la Ang II, la Ang II exogena no modifico la agregacion plaquetaria inducida por U46619. La union del U46619 a las plaquetas fue competitivamente inhibida por el losartan en forma dependiente de la dosis. Sin embargo, solo dosis altas de EXP 3174 redujeron la union del U46619. Captopril no modifico la union del U46619 a las plaquetas. Conclusiones Losartan disminuyo la agregacion plaquetaria por un mecanismo dependiente de TXA2. El EXP 3174 demostro una menor potencia que losartan en reducir la activacion plaquetaria por TXA2. El captopril y la Ang II exogena no tuvieron efecto sobre la activacion de plaquetas humanas. Estos resultados sugieren que el losartan redujo la activacion plaquetaria inducida por el TXA2 por un mecanismo independiente del bloqueo de los receptores de tipo AT-1.

Effect of losartan on human platelet activation

OBJECTIVE Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. METHODS Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). RESULTS U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. CONCLUSIONS Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.

Doxazosin modifies Bcl-2 and Bax protein expression in the left ventricle of spontaneously hypertensive rats

Background Increased apoptosis has recently been reported in the heart of spontaneously hypertensive rats (SHRs). Objective To investigate the molecular basis of apoptosis in the left ventricle of SHRs in terms of the expression of Bcl-2 protein (which protects from apoptosis) and Bax protein (which acts as an apoptotic promoter). In addition, we analysed the involvement of α1-adrenergic receptors in the left ventricular apoptosis of SHRs. Methods The study was performed in untreated SHRs (n = 16) and SHRs that were orally treated with doxazosin (10 mg/kg body weight per day, for 15 days), a selective α1-receptor blocker (n = 16). A group of Wistar–Kyoto (WKY) rats (n = 16) was used as the control. Results The left ventricles of untreated SHRs showed a significant increase in Bcl-2 protein expression and a reduced presence of Bax protein. The ratio of Bcl-2:Bax in SHRs was higher than in WKY rats, suggesting an anti-apoptotic state. Paradoxically, both the number of apoptotic cardiac cells and the cleavage of an 85-kDa fragment of the poly (ADP-ribose) polymerase (PARP), a marker of caspase-3 activity, were higher in the left ventricle of SHRs than in WKY rats, suggesting an apoptotic situation. Bax promotes cell apoptosis when it is bound to Bcl-2. We then determined the abundance of Bax–Bcl-2 complexes in the left ventricle of the two groups of animals. Bax–Bcl-2 complexes were more abundant in SHRs than WKY rats. In a second set of experiments, we analysed the role of α1-adrenergic blockade by doxazosin in the above-described mechanisms. Doxazosin treatment reduced the formation of Bax–Bcl-2 complexes in the left ventricle of SHRs, and this was accompanied by a decrease in the levels of 85-kDa PARP and a reduction in apoptotic left ventricular cells. Conclusions The present work suggests that the presence of Bax–Bcl-2 complexes in the left ventricle could be a more reliable marker of the apoptotic state than the determination of the absolute expression of Bcl-2 and Bax proteins. Moreover, the inhibition of α1-adrenergic receptors by doxazosin decreased the abundance of Bax–Bcl-2 complexes and promoted a reduction of apoptosis in the left ventricle of SHRs.